Transgenic algae for delivering antigens to an animal

ABSTRACT

Delivery systems and methods are provided for delivering a biologically active protein to a host animal. The systems and methods provided include obtaining an algal cell transformed by an expression vector, the expression vector comprising a nucleotide sequence coding for the biologically active protein, operably linked to a promoter. In one illustrated embodiment, the biologically active protein is an antigenic epitope and upon administration to the animal the algal cell induces an immune response in the host animal.

FIELD OF THE INVENTION

[0001] This invention relates to a system for delivering antigens to ananimal.

BACKGROUND OF THE INVENTION

[0002] Proteins, dipeptide and polypeptide (hereinafter collectivelyreferred to as proteins) are responsible for most of the activities of acell, such as catalysis, communication, defense, movement, andtransport.

[0003] Proteins can be delivered to animals for the purpose ofactivating or supplementing a biological activity. Examples includeantigens that activate an immune response and hormones that regulategrowth and development.

[0004] The bases of a protein's biological activity is its sequenceand/or conformation. Hence, the biologically active portion (such as anepitome) should remain essentially intact until it reaches its targetdestination. Factors that could limit the biological activity of aprotein include chemical and enzymatic denaturation, as well asstructural barriers that preclude entry into the animal or access to thetarget destination.

[0005] The patent describes a method for producing, and then deliveringbiologically active proteins to an animal using transgenic algae. Thedelivery of an antigen to an animal and activation of an immune responseis offered as a specific example.

[0006] Infectious Disease in Humans

[0007] Infectious disease is an age-old problem. Early in human history,infectious diseases such as smallpox, bubonic plague, influenza, measlesand many others caused epidemics and countless deaths. More recently,epidemics of Legionnaires' disease, human immunodeficiency virus, Ebolavirus, Lyme disease, and others have been significant threats to humanhealth.

[0008] Solutions to Infectious Disease

[0009] Early in human history, quarantine of infected individuals andimproved sanitation measures were used to decrease the spread ofinfectious disease. Later, chemotherapeutic agents (drugs) were inventedand, early on, included chemicals like sulfur and mercury. Modernchemotherapeutics include antibiotics, antiviral drugs, andantiparasitic drugs. Although essential, there are properties of suchdrugs that are not ideal. For example, drugs do not prevent disease.Rather, drugs are administered after a disease is diagnosed. Anotherproblem is that infectious agents can develop resistance to the drugssuch that the drugs are no longer effective against the infectiousagents. Finally, drugs can cause serious side affects in the individualto which they are administered.

[0010] An alternative to chemotherapeutic agents is administration ofimmunogenic agents, wherein the immunogens that comprise a compositionoriginating from the infectious agent whose infection one is trying toprevent. After administration, such immunogenic compositions preferablystimulate the immune system such that subsequent infection by theinfectious agent is prevented, or disease symptoms caused by theinfectious agent are decreased. Such immunogenic compositions areadvantageous in that they are administered before the individualcontracts the infectious agent. The stimulation of immunity in theindividual caused by administration of the immunogenic composition,therefore, is designed to prevent infection and disease. Suchimmunogenic compositions normally produce few side effects in theindividual to which the composition is administered. Finally, infectiousagents do not normally develop resistance to immunity that develops as aresult of administration of the antigen composition.

[0011] Infectious Disease in Non-Human Animals

[0012] Infectious diseases in non-human animals also cause significantmorbidity and mortality. Such disease is important, not only becausenon-human animals can sometimes transmit the infectious agents tohumans, but also because non-human animals and the products thereof areimportant human food sources and their loss is economically burdensome.

[0013] Infectious Diseases in Aquaculture

[0014] An example of an area where infectious non-human animal diseasescontinue to affect an important human food source is aquaculture.Aquaculture is the fanning of aquatic organisms, including fish andother seafood, for human consumption. Currently in the U.S., thedomestic fishing industry meets only a small part of the total demandfor fish. In 1997, for example, the federal trade deficit for importedfish was nearly $9 billion, the third largest component of the U.S.foreign trade deficit.

[0015] In an attempt to meet the demand for fish, the aquacultureindustry has responded and, in 1997, produced over $55 billion in farmedfish (statistic from the Food and Agriculture Organization of the UnitedNations). Furthermore, the aquaculture industry has grown, historicallybetween 10-20% per year for the last ten years. Therefore, it is clearthat aquaculture is a rapidly emerging supplement and replacement forthe traditional fishing industry and has tremendous growth potential.

[0016] One of the major constraints for aquaculture, however, isdisease. Under the high density conditions under which fish and otheraquatic organisms are farmed, the incidence of infectious disease can behigh and, when disease does occur, it can spread rapidly through entirepopulations with high mortality. On average, 10-30% of farmed fishproduction, and up to 80% of shrimp production, is lost due to disease(Austin B., et al., 1987 Bacterial Fish Pathogens: Disease in Farmed andWild Fish, 364 pp Publishers: (Ellis Horwood Ltd., Chichester, UK)).

[0017] Solutions to Infectious Diseases in Aquaculture

[0018] Again using aquaculture as a specific example, fish diseases witha bacterial etiology can be effectively treated with chemotherapeuticagents from the class called antibiotics. However, as much as 80% of theantibiotic may pass through the fish (Pothuluri, et al.,1998, Res. Dev.Microbiol 2:351-372), and development of bacteria resistant to theantibiotic may also arise. Such antibiotic-resistant bacterial pathogenscan spread, creating entire fish populations harboring pathogenicbacteria that are resistant to the antibiotic. Clearly, it would beadvantageous to prevent infection of the fish by the bacteriaaltogether. Another consideration is that viral and parasitic diseasescannot be treated with antibiotics.

[0019] Another strategy for preventing infection and reducing fishlosses due to disease is prophylactic administration of an antigencomposition, wherein the antigens are derived from an infectious agent,to stimulate the immune system of fish, other aquatic organisms, orother organisms generally (Gudding, et al.,1999, Vet. Immun. Immunopath.72: 203-212).

[0020] Methods of Introducing Antigens into Animals

[0021] One problem with antigen compositions, especially in fish, isthat many methods for administering them may not be technically oreconomically practical. For example, direct injection of the antigencomposition into fish is labor intensive and is often expensive relativeto the future market value of the fish. Furthermore, injection needlescan cross-infect fish with contaminating infectious agents, andaccidental injection of humans can cause severe infections andanaphylactic reactions. In addition, noninjurious injection of smallfish may be difficult.

[0022] An alternative route of administration is an oral method whereinthe antigen composition is incorporated into the fish food, for example.Another improved method of administering antigens to fish is immersionof the fish for a preset period of time in a suspension of the antigen.However, it can be costly to produce, purify, and package the antigensfor such use. Prior art methods of producing antigens have involved thedifficulty of growing fish viruses in culture systems to produce enoughvirus to obtain a sufficient quantity of antigen. In addition, antigencompositions to be administered orally are often encapsulated inexpensive polysaccharide-coated beads. Finally, oral administration ofantigen compositions have previously shown low and inconsistent levelsof stimulation of the fish immune system, thereby minimizing protectionagainst subsequent infection by the infectious agent.

SUMMARY OF THE INVENTION

[0023] In accordance with the present invention, a delivery system isprovided for delivering peptides to a host animal. The peptides may begrowth hormones, antigens derived from a pathogen, other antimicrobialpeptides, etc. The delivery system is a transgenic algae that comprisesa transgene which comprises a) a polynucleotide encoding at least onepeptide, for example an antigenic determinant for the pathogen, and b) apromoter for driving expression of the polynucleotide in the algae. In apreferred embodiment, the transgene further comprises c) a terminatorthat terminates transcription, and d) all other genetic elementsrequired for transcription. In another preferred embodiment, thetransgenic algae further expresses the peptide. If the peptide is anantigenic determinant it is preferably located on the cell surface orwithin the cytoplasm or an organelle of the transgenic algae. Thetransgenic algae of the present invention is useful for inducing animmune response in the host animal to the pathogen. The transgenic algaeof the present invention is also for treating, ameliorating, orpreventing a disease caused by the pathogen.

[0024] The present invention also provides methods for delivering thepeptide, for example, an antigenic determinant derived from a pathogen,to a host animal. In one aspect, the method comprises orallyadministering a transgenic algae which comprises (a) a polynucleotidethat encodes at least one antigenic determinant of a pathogen for thehost animal and (b) a promoter that drives expression of thepolynucleotide in the nucleus or an organelle of the algae. Such methodis especially useful for delivering the antigenic determinant to amammal or an aquatic animal. In another aspect, the method comprisesimmersing the host animal into a suspension comprising water and atransgenic algae which comprises (a) a polynucleotide that encodes atleast one antigenic determinant of a pathogen for the host animal, and(b) a promoter that drives expression of the polynucleotide in thenucleus or an organelle of the algae. Such method is especially usefulfor delivering the antigenic determinant to an aquatic animal.

[0025] Thus, in one aspect of the invention, a delivery system isprovided for delivering a biologically active protein to a host animalcomprising an algal cell transformed by an expression vector, theexpression vector comprising a nucleotide sequence coding for thebiologically active protein, operably linked to a promoter.

[0026] In another aspect of the invention, a delivery system is providedfor delivering antigens to a host animal comprising an algal celltransformed by an expression vector, the expression vector comprising anucleotide sequence coding for an antigenic determinant.

[0027] In still another aspect of the invention, a method is providedfor inducing an immune response in an animal, comprising the steps ofobtaining a transgenic alga expressing an antigenic peptide,administering the transgenic alga to the animal.

[0028] Additional features of the present invention will become apparentto those skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE FIGURES

[0029] The present invention may be more readily understood by referenceto the following figures wherein:

[0030]FIG. 1 shows the pSSCR7 plasmid used for constructing nucleartransfection vectors for use in the present invention;

[0031]FIG. 2 shows the pCPPTG plasmid used for constructing chloroplasttransfection vectors for use in the present invention;

[0032]FIG. 3 is a diagram showing the expression between the 3′ end of alow CO₂-induced plasma-membrane protein gene and a P57 antigen, andshowing that the P57 antigen is located on the periplasm side of thecell membrane;

[0033]FIG. 4 is a western blot of trout sera probed against algal cellsused for oral immunization. Lane 1: E-22; Lane 2: CP57; Lane 3:CC-2137(WT); Lane 4: E-22; Lane 5: CP57; Lane 6: CC-2137(WT); Lane 7:E-22; Lane 8: CP57; Lane 9: CC-2137(WT); Lane 10: E-22; Lane 11: CP-57;Lane 12: CC-2137(WT); and

[0034]FIG. 5 is a western blot of Trout Mucus Probed Against Algal Cellsused for Immersion Immunization. Lane 1: E-22; Lane 2: CP57; Lane 3:CC-2137(WT); Lane 4: E-22; Lane 5: CP57; Lane 6: CC-2137(WT); Lane 7:E-22; Lane 8: CP57; Lane 9: CC-2137(WT); Lane 10: E-22; Lane 11: CP-57;Lane 12: CC-2137(WT).

DETAILED DESCRIPTION OF THE INVENTION

[0035] In one aspect of the present invention provides a delivery systemfor introducing one or more peptides into a host animal. The deliverysystem involves the use of is a transgenic algae comprising a transgenewhich comprises a polynucleotide encoding one or more peptides and apromoter which drives expression of the peptide encoding sequence in thealgae.

[0036] Antigenic Determinant

[0037] The term “antigenic determinant” as used herein refers to aprotein or polypeptide which is capable of eliciting an immune responseor defense response in the host animal. The antigenic determinant is atleast partially derived from a pathogenic microorganism. Herein, theterm “microorganism” means a bacterium, virus, fungus, or parasite(i.e., protozoan or helminth, for example). Preferably, the immuneresponse or defense response elicited by the antigenic determinant isprotective in the host animal in the sense that a subsequent infectionof the host animal by the pathogenic organism from which the antigenicdeterminant is derived would be prevented, would not cause disease or,if disease were caused, the disease or symptoms associated with thedisease would be ameliorated. Preferably, the antigenic determinantitself does not cause disease or any other adverse symptoms in the hostanimal.

[0038] The antigenic determinant is either a holoprotein or a portion ofa protein that stimulates the immune system of an animal that is a hostfor the pathogenic organism. Typically, the antigenic determinants areeither secreted by the pathogen or is found on the cell membrane or cellwall thereof, but could potentially be any component of the pathogen.The antigenic determinant may be part of fusion protein. For example, itmay be advantageous to fuse the antigenic determinant with a proteinthat is expressed on the surface of the algal cell.

[0039] The transgenic algae of the present invention comprise at leastone antigenic determinant of a pathogenic microorganism. In certainembodiments the present transgenic algae comprise a plurality ofantigenic determinants from a single microorganism. In otherembodiments, the present transgenic algae comprise one or more antigenicdeterminants from a plurality of pathogenic microorganisms. Whenexpressed, the antigenic determinants may be located in the cytoplasm,in an organelle, particularly the mitochondria or chloroplast, on thecell surface, exported from the cell or in a combination of locations inthe algae.

[0040] Host Animals

[0041] As used herein the term “host animal” refers to all animalscapable of mounting an immune or defense response when infected with apathogenic microorganism. Accordingly, the term host animal, as usedherein, encompasses mammals, including humans, companion animals such ascats and dogs, non-companion animals such as cattle and sheep, birds,aquatic vertebrates, and aquatic invertebrates.

[0042] Because algae are a food substance for numerous aquatic animals,the transgenic algae of the present invention are especially useful fordelivering an antigenic determinant to aquatic vertebrates andinvertebrates. Such aquatic vertebrates include, but are not limited to,all vertebrate fish, which may be bony or cartilaginous fish. Suchfin-fish include but are not limited to salmonids, carp, catfish,yellowtail, seabream, and seabass.

[0043] Immune systems in fish are essentially the same as the immunesystems of mammals. The immune system is organized into discretecompartments to provide the milieu for the development and maintenanceof effective immunity. Those two overlapping compartments: the lymphoidand reticuloendothelial systems (RES) house the principal immunologiccells, the leukocytes. Leukocytes derived from pluripotent stem cells inthe bone marrow during postnatal life include neutrophils, eosinophils,basophils, monocytes and macrophages, natural killer (NK) cells, and Tand B lymphocytes. Hematopoietic and lymphoid precursor cells arederived from pluripotent stem cells. Cells that are specificallycommitted to each type of leukocyte (colony-forming units) areconsequently produced with the assistance of special stimulating factors(e.g., cytokines).

[0044] Leukocytes, the main cells in the immune system, provide eitherinnate or specific adaptive immunity. These cells are derived frommyeloid or lymphoid lineage. Myeloid cells include highly phagocytic,motile neutrophils, monocytes, and macrophages that provide a first lineof defense against most pathogens. The other myeloid cells, includingeosinophils, basophils, and their tissue counterparts, mast cells, areinvolved in defense against parasites and in the genesis of allergicreactions. In contrast, lymphocytes regulate the action of otherleukocytes and generate specific immune responses that prevent chronicor recurrent infections.

[0045] Lymphoid Cells provide efficient, specific and long-lastingimmunity against microbes and are responsible for acquired immunity.Lymphocytes differentiate into three separate lines: thymic-dependentcells or T lymphocytes that operate in cellular and humoral immunity, Blymphocytes that differentiate into plasma cells to secrete antibodies,and natural killer (NK) cells. T and B lymphocytes are the only lymphoidcells that produce and express specific receptors for antigens. TLymphocytes are involved in the regulation of the immune response and incell mediated immunity and help B cells to produce antibody (humoralimmunity). Mature T cells express antigen-specific T cell receptors(TcR) that are clonally segregated (i.e., one cell lineage-one receptorspecificity). Every mature T cell also expresses the CD3 molecule, whichis associated with the TcR. In addition mature T cells display one oftwo accessory molecules, CD4 or CD8. The TcR/CD3 complex recognizesantigens associated with the major histocompatibility complex (MHC)molecules on target cells (e.g. virus-infected cell). The TcR is atransmembrane heterodimer composed of two polypeptide chains (usually,alpha and beta chains). Each chain consists of a constant (C) and avariable (V) region, and is formed by a gene-sorting mechanism similarto that found in antibody formation. The repertoire is generated bycombinatorial joining of variable (V), joining (J), and diversity (D)genes, and by N region (nucleotides inserted by the enzymedeoxynucleotidyl-transferase) diversification. Unlike immunoglobulingenes, genes encoding TcR do not undergo somatic mutation. Thus there isno change in the affinity of the TcR during activation, differentiation,and expansion.

[0046] The activation of B cells into antibody producing/secreting cells(plasma cells) is antigen-dependent. Once specific antigen binds tosurface Ig molecule, the B cells differentiate into plasma cells thatproduce and secrete antibodies of the same antigen-binding specificity.If B cells also interact with Th cells, they proliferate and switch theisotype (class) of immunoglobulin that is produced, while retaining thesame antigen-binding specificity. This occurs as a result ofrecombination of the same Ig VDJ genes (the variable region of the Ig)with a different constant (C) region gene such as IgG. In the case ofprotein antigens, Th2 cells are thought to be required for switchingfrom IgM to IgG, IgA, or IgE isotypes. IgM is therefore the principalantibody produced during a primary immunization. This primary antibodyresponse is manifested by serum IgM antibodies as early as 3-5 daysafter the first exposure to an immunogen (immunizing antigen), peaks in10 days, and persists for some weeks. Secondary or anamnestic antibodyresponses following repeated exposures to the same antigen appear morerapidly, are of longer duration, have higher affinity, and principallyare IgG molecules.

[0047] When antibodies bind to antigens, they may 1) neutralizepathogenic features of antigens such as their toxins, 2) facilitatetheir ingestion by phagocytic cells (opsonization), 3) fix to andactivate complement molecules to produce opsonins and chemoattractants(vide infra), or 4) participate in antibody-dependent cellularcytotoxicity (ADCC). In addition to antibody formation, B cells alsoprocess and present protein antigens. After the antigen is internalizedit is digested into fragments, some of which are complexed with MHCclass II molecules and then presented on the cell surface to CD4+ Tcells.

[0048] In the case of aquatic vertebrates, examples of pathogenicmicroorganisms whose antigenic determinants may be expressed in thetransgenic algae include, but are not limited to Rennibacteriumsalmoninarum (causative agent of bacterial kidney disease in salmon,trout, char and whitefish; i.e., salmonids), Aeromonas salmonicida,Aeromonas hydrophila, species of Vibrio (including V. anguillarum and V.ordalii) species of Pasteurella (including P. piscicida), species ofYersinia, species of Streptococcus, Edwardsiella tarda and Edwardsiellaictaluria; the viruses causing viral hemorrhagic septicemia, infectiouspancreatic necrosis, viremia of carp, infectious hematopoietic necrosisvirus, channel catfish virus, grass carp hemorrhagic virus, nodaviridaesuch as nervous necrosis virus or striped jack nervous necrosis virus,infectious salmon anaemia virus; and the parasites Ceratomyxa shasta,Ichthyophthirius multifillius, Cryptobia salmositica, Lepeophtheriussalmonis, Tetrahymena species, Trichodina species and Epistylus species.

[0049] Examples of proteins for inducing an immune response in certainaquatic vertebrates include but are not limited to p57 leukocyteagglutinizing protein from Rennibacterium salmoninarum and the G-proteinfrom infectious hematopoietic necrosis virus (IHNV).

[0050] Aquatic invertebrates which are suitable host animals include,but are not limited to, shrimp, crabs, oysters, and clams.

[0051] Shellfish possess a defense system capable of defending againstpathogens. This system has many similarities to the nonspecific defensesystem of vertebrates, such as the activation of phagocytotic cells(hematocytes). However, there is no evidence that shrimp or otherinvertebrates possess a specific defense response similar to the immunesystem of vertebrates. Other defense responses activated followinginfection reported for shrimp include: increases in hematocyteproduction, production of active oxygen species, activation ofphenoloxidase and subsequent melanin synthesis, the production ofantimicrobial peptides, encapsulation of the foreign body andcoagulation of hematocytes (Rodriquez, et al.2000, Aquaculture172:351-372). Unique to invertebrates are specific proteins, such as abeta glucan-binding protein (BGBP) and a lipopolysaccharride-bindingagglutinin (LSBA), that recognize cell-wall components of microorganismsand subsequently mobilize hematocytes for phagocytosis (Bachere, E.,2000, Aquiculture 191: 3-11).

[0052] Invertebrates have many similarities to the defense response inplants, such as the production of phenolic compounds and reactive oxygenspecies and the mechanism in which the defense system is activated. Theplant-pathogen interaction is a well-characterized signal transductionsystem composed of nonspecific and specific pathogen elicitors—many theproduct of an avirulence gene (avr)—and their cognate plant receptors.Following receptor recognition of an elicitor, intracellular mediatorsorchestrate a cascade response that activates several well-characterizeddefense mechanisms and, is some cases, short-term acquired immunitymediated by systemically transported signaling molecules. BGBP and LSBAhave an analogous role in orchestrating a defense cascade in shrimp thatalso includes a short-term acquired immunity.

[0053] Numerous signaling molecules can activate the defense system ofinvertebrates, most of which are of pathogen origin. Treatment with deadbacteria or cell wall components, such as B-glucans,lippopolysaccarides, and peptoglycans, have been reported to provideprotection against shrimp pathogens when administered prior tochallenge. (Alabi, et al. 1999, Aquaculture 178:1-11) reported thatfresh or freeze dried Vibrio harveyi administered by immersion, but notorally, reduced infection of P. indicus protozoa by V. harveyi for 48hours. More virulent strains increased the level of protectionsuggesting that the defense response is activated either by differentdefense eliciting molecules (qualitative) or different numbers of thesame defense eliciting molecules(quantitative). Furthermore, testedstrains provided cross-protection suggesting that this is a non-specificresponse. Others have reported that peptoglycan extracted from the cellwall of the nonpathogen Bifidobacterium thermophilium, providedprotection against vibrosis and white spot syndrome baculovirus whenadministered orally. These results further support that invertebratesrespond to pathogens through a general nonspecific mechanism that can beactivated by different elicitor molecules and is effective against abroad range of pathogens. These defense-activating signaling molecules(elicitors) do not result in the production of antibodies. However, forpurposes of convenience, the term “antigenic determinant” as used hereinalso encompasses the elicitors which prompt a defense response inaquatic invertebrates.

[0054] Examples of microorganisms which are pathogenic for aquaticinvertebrates include, but are not limited to, white spot syndrome virus(WSSV), tuarus virus, IHHNV and Vibrio harveyii.

[0055] Examples of proteins for inducing an immune response ininvertebrates include, but are not limited to, the VP28, VP26c and VP24proteins of WSSV shrimp virus. See van Hulten et al., Virology266:227-236 (2000) and WO 0109340 for a discussion of WSSV and its usein vaccines for crustaceans.

[0056] Algae

[0057] Algae are plant-like organisms without roots, stems or leaves.Algae are distinct from plants in several ways, includingphylogenetically, biochemically, and morphologically. Algae containchlorophyll and vary in size from microscopic forms (phytoplankton) tomassive seaweeds. Their habitat is fresh or salt water, or moist places.Most algae are eukaryotic (sub-kingdom=phyciobionta), but several (e.g.,cyanobacteria and prochlorophyta) are prokaryotic.

[0058] The transgenic algae of the present invention encompass bothprokaryotic and eukaryotic algae, which preferably are unicellular.Unicellular algae are also known as microalgae. Preferably, the presenttransgenic of the present invention algae comprise walls that eitherlack pores or gaps (referred to hereinafter as “walled” algae) or thatcontain pores or gaps (referred to hereinafter as “wall-less” algae).Optionally, the wall-less algae are coated with a polysaccharide polymeras described in (Tsai, et al., 1994, Progress. Fish-Culturist 56: 7-12)

[0059] The transgenic algae of the present invention comprise atransgene comprising an exogenous polynucleotide which encodes theantigenic determinant and is operably linked to a promoter which drivesexpression of the exogenous polynucleotide in the algae, and preferablycomprises other genetic elements required for expression. Preferably,the transgene comprises a terminator for ending transcription. Alsopreferably, the transgene is stably integrated into the genomic materialfound in the nucleus (known hereinafter as a nuclear transformant), thechloroplast (known herein after as a chloroplast transformant), ormitochondria (known hereinafter as a mitochondrial transformant’) of thealgae. In nuclear transformants, the antigenic determinants preferablyare expressed on the cell membrane or cell wall of the transgenic algaeor in the cytoplasm thereof.

[0060] In certain preferred embodiments, the algae is a green algae(Chlorophyta), a brown algae ((Phaeophyta), or diatoms(Bacillariophyta).

[0061] Examples of green algae, which are especially well-suited for useas the delivery system, include members of the Chlamydomonas species,particularly Chlamydomonas reinhardtii; the Chlorella species, theVolvox species, and some marine macrophytes. Chlamydomonas reinhardtii,a unicellular eukaryotic green algae is particularly advantageous foruse in introducing antigens into animals. C. reinhardtii growvegetatively through mitotic division of haploid cells. Haploid cellsare of either the (−) or (+) mating type. When grown in the absence ofnitrogen, haploid cells of opposite mating types associate, are heldtogether through their flagella, and eventually fuse to form a diploidzygospore. The diploid zygote undergoes meiosis and releases fourhaploid cells that resume the vegetative life cycle. One example of awalled green algae is Chlamydomonas strain CC-744. One example of awall-less green algae is Chlamydomonas strain CC-425. Both of thesestrains are available from Chlamydomonas Genetic Stock Center, DukeUniversity (Durham, N.C.).

[0062]Chlamydomonas reinhardtii is particularly preferred because itgrows rapidly and is easily and inexpensively grown in culture.Exogenous DNA can easily be introduced into the nuclear, chloroplast,and mitochondrial genome of this algae, and can be expressed at highefficiency (≧1% of total cellular protein). Auxotrophic mutants (mutantsthat differ from the wild-type in requiring one or more nutritionalsupplements for growth) are readily available at the ChlamydomonasGenetic Stock Center. Such mutants can be genetically complemented bytransformed DNA (i.e., exogenous DNA introduced into the cell), whichfacilitates selection of algae containing a desired transgene. Thisselection method is preferred, at it is free from use of antibiotics.

[0063] In addition to the ease of growth and genetic manipulation, ascited above, there are additional characteristics of C. reinhardtii thatmake the organism useful for delivering antigens to animals. C.reinhardtii is a potential food source for animals, especially larvalfish and marine invertebrates (C. reinhardtii is nontoxic andnonpathogenic. Both freshwater C. reinhardtii and a related marinespecies, C. pulsatilla, are available for administering antigens toaquatic organisms in both environments.

[0064] Optionally, the algae of the present invention are geneticallyengineered such that they will not proliferate unless they are in veryspecific controlled environments (i.e., such strains will not grow ortransfer their genes in the wild). Within the context of thisapplication, such algae are said to be “disabled.” Use of such disabledstrains inhibits or limits spread of the transgenic algae of the presentinvention into the environment.

[0065] Such disabled strains of algae, particularly strains of C.reinhardtii, are constructed by incorporating into the genomes of suchstrains various genetic mutations that preclude growth and/or matingoutside of a specific environment. For example, the transgenic algae maybe engineered to contain mutations that prevent photosynthesis. Strainscontaining such mutations are unlikely to survive in the wild becausethey cannot produce the energy or reduced carbon necessary to sustainlife. Such mutant-containing strains, however, can be grown in thelaboratory using acetate as a carbon source. One such type of mutationpreventing photosynthesis occurs in, but is not limited to, genescomprising the psbD/psaC operon of C. reinhardtii, which is part of thechloroplast genome of the organism. Such mutations in the chloroplastgenome are preferably in the chloroplast genome of cells of the (+)mating type. C. reinhardtii of the (−) mating type generally do notsurvive after a mating to produce a diploid cell has occurred (seebelow).

[0066] Other mutations useful in constructing disabled algal strains aremutations in genes resulting in strains that cannot grow in the absenceof specific metabolites (i.e., substances produced by, or taking partin, metabolism). Such strains are said to be “auxotrophic” for thatparticular metabolite. Strains auxotrophic for various amino acids,vitamins, nucleotides, and so forth are particularly useful. Forexample, some such useful mutations require cells to be grown in thepresence of arginine, thiamine, or nicotinamide.

[0067] Other mutations that affect the ability of algae to grow and/ortransfer its genes, although such mutations are not specifically statedherein, are also included within the scope of this invention.

[0068] In one embodiment of this invention multiple mutations of thetype described above, for example, are combined into a single strain ofalgae. Combinations of these mutations in a single strain (also called“stacking” of mutations) result in disabled strains that areparticularly nonfunctional in growth and mating. Such strains of algaeare particularly unable to grow and transfer their genes in the wild.

[0069] Another strategy useful in making disabled algal strains embodiedin this invention takes advantage of events that occur naturally in amating event. In C. reinhardtii, when haploid (−) and (+) cells mate toform a diploid cell, only the chloroplast genomes from the (+) matingtype organism survive. The chloroplast genomes of (−) mating type cellsare degraded during mating. Therefore, C. reinhardtii strains in whichthe transgenes encoding the antigenic determinant are located in thechloroplast genome of a (−) cell are advantageous when control ofproliferation of transgenic algae is desired.

[0070] Another type of disabled algal strain that is included in thisinvention are algal strains that have mutations in the genes encodingflagella. For example, in C. reinhardtii, flagella are necessary to hold(−) and (+) cells together so that mating can occur. Therefore, certaintransgene-containing cells with mutations in genes encoding flagellawill be unable to transmit the transgene through mating.

[0071] An additional disabling strategy that is part of the presentinvention is use of the freshwater alga, C. reinhardtii, for use intransferring antigens into saltwater aquatic organisms, as C.reinhardtii is unable to survive in seawater for more than an hour. C.reinhardti strains containing the P5CS gene for proline synthesis canalso be used similarly when survival of the algae for longer times isimportant for a particular application. Strains containing the P5CS genecan tolerate seawater for up to 10 hours before 100% mortality occurs.

[0072] Preparation of the Transgenic Algae

[0073] In addition to an exogenous polynucleotide encoding an antigenicdeterminant of a pathogenic microorganism, the transgene which isincorporated into the transgenic algae comprises a promoter whichregulates transcription of the exogenous gene in the nucleus,chloroplast, or mitochondria, of the algae, and preferably other geneticelements required for expression. The transgene also preferably includesa terminator for terminating transcription.

[0074] To prepare vectors for making the transgenic algae, the exogenouspolynucleotide encoding the antigenic determinant is first cloned intoan expression vector, a plasmid that can integrate into the algalgenome. In such an expression vector, the DNA sequence which encodes theantigenic determinant or a fusion protein comprising the antigenicdeterminant is operatively linked to an expression control sequence,i.e., a promoter, which directs mRNA synthesis. Preferably, the promoteris an endogenous promoter, i.e., it directs transcription of genes thatare normally present in the algae. Examples of suitable promoters forChlamydomonas reinhardtii include, but are not limited to, thechloroplast gene promoter psbA and the nuclear promoter region of the β₂tubulin gene. The expression vector, may also contains a ribosomebinding site for translation initiation and a transcription terminator.Preferably, the recombinant expression vector also includes an E. coliorigin of replication and an E. coli selectable marker to facilitatecloning of the vector in E. coli.

[0075] In one embodiment, the exogenous polynucleotide encoding theanitigenic determinant is fused to a polynucleotide which encodes amembrane protein to express the antigen on the surface of the cell. Forexample, the predicted folding topology of the CO₂-induced surfaceprotein of Chlamydomonas reinhardtii indicates that both the N-terminusand the C-terminus are located on the periplasmic surface of the plasmamembrane. Thus, this protein is especially useful for expressing theantigenic determinant on the plasma membrane of Chlamydomonasreinhardti.

[0076] Plasmids are introduced into the algae by standard transformationmethods known to those skilled in the art, such as for example,electroporation, vortexing cells in the presence of exogenous DNA, acidwashed beads, polyethylene glycol, and biolistics.

[0077] One particular advantage of the present invention is that genesfor multiple antigens from one infections agent, or multiple antigensfrom different infectious agents, can be expressed simultaneously in asingle algal cell. Such multiple genes or epitopes can be included in asingle vector, for example a plasmid, or in multiple plasmids, each ofwhich must be transformed into the algae. The advantage of such an algaethat expresses multiple antigens, is that all of the antigens areintroduced into the animal by administration of the particular algalstrain to the animal.

[0078] Transformation of the algae is determined by assaying for thepresence of the gene encoding the antigen by PCP, for example.Procedures known to those of skill in the art, such as for example,deflagellation, copper addition, and ammonium addition of the algae, maybe used to enhance expression of the antigenic determinant in thetransgenic algae. The choice of such procedure depends upon the promoterused to prepare the construct. See, for example, Davies et al., NucleicAcids Research 20: 2959-2865 (1992); Dutcher, Current Opinions in CellBiology 13:39-54 (2001); Moseley et al., Photosynthesis: Mechanisms andEffects.

[0079] One type of plasmid vector integrates into the nucleus of algalcells and expresses its proteins which are localized to the cytoplasm ofalgal cells. One particular vector of this type is pSSCR7, derived froma the plasmid described in Davies (Davies et al. (1994) Plant Cell6:53-63).

[0080] Another type of vector also integrates into the nucleus butexpresses proteins that are localized to the periplasm. One particularvector of this type is a derivative of pSSCR7 which has a 5′ arylsulfatase periplasmic targeting transit sequence (Davies et al. (1994)Plant Cell 6:53-63).

[0081] A third type of vector integrates into the chloroplast genome byhomologous recombination and expresses proteins that are localized tothe chloroplasts (Hutchinson, et al., 1996, Chapter 9, Chloroplasttransformation. Pgs. 180-196; In: Molecular Genetics of Photosynthesis,Frontiers in Molecular Biology. Anderson B., Salter A H, and Barber J.eds.: Oxford University Press).

[0082] Administration of Transgenic Algae to Animals

[0083] Animals to which the transgenic algae are administered include,but are not limited to, mammals, birds, and aquaculture species.Aquaculture species include a diversity of species of cultured fin-fish,shellfish, and other aquatic animals. Fin-fish include all vertebratefish, which may be bony or cartilaginous fish. Such fin-fish include butare not limited to salmonids, carp, catfish, yellowtail, seabream, andseabass. Salmonids are a family of fin-fish which include trout(including rainbow trout), salmon, and Arctic char. Examples ofshellfish include, but are not limited to, clams, lobster, shrimp, crab,and oysters. Other cultured aquatic animals include, but are not limitedto eels, squid, and octopi.

[0084] One method of administering the present transgenic algae toanimals is oral delivery of the algae to the animal by feeding. Thetransgenic algae may be delivered to the animal as a dried cell powderor as a component of the normal diet. For example, in one method offeeding the algae to fish, up to 5% freeze-dried transgenic algae areadded to an aqueous mixture containing 5% of a casein-gelatin basedprotein source. The ingredients are cold-pelleted, freeze-dried, crushedinto 2 mm particles, and fed to the fish. Live algae could be deliveredin gelatin capsules.

[0085] Another method of administering algae to animals, particularlyaquatic animals, is immersion of the host animal in a suspension of livealgae. Good results have been obtained by immersing trout in an aqueoussuspension containing from between 10⁵-10⁷ algae per ml of water. Fishwere immersed in the suspension anywhere from between 20 seconds to 2hours, and then removed from the suspension. The immersion method isparticularly advantageous for introducing algae into smaller fish (lessthan about 10 to 15 grams in weight). Such method can be used incombination with oral delivery of algae though feeding, as describedabove. Other methods of delivery are possible and are within the scopeof this invention.

[0086] The transgenic algae are introduced into the animal in a regimendetermined as appropriate by a person skilled in the art. For example,the transgenic algae may be introduced into the animal multiple times(e.g., two to five times) at an appropriate interval (e.g., every two tothree weeks) and dosage or dilution, by normal feeding or by immersion.

EXAMPLES

[0087] The following examples are for purposes of illustration only andare not intended to limit the scope of the invention as defined in theclaims which are appended hereto. The references cited in this documentare specifically incorporated herein by reference.

Example 1 Transgenic Algae Expressing the P57 Immunogen fromRennibacterium salmoninarum

[0088]Rennibacterium salmoninarum is the etiologic agent for bacterialkidney disease, the most common disease of farmed salmonoids. Atransgenic algae expressing an antigenic determinant of the fishpathogen. R.salmoninarum was prepared using a portion of the P57leucocyte agglutinizing protein (Genbank accession no. AF123889) ofRennibacterium salmoninarum for surface display as an antigen.

[0089] A highly antigenic determinant of the P57 protein encoding theamino acids VYNKDGPAKELKV, (SEQ ID NO: 1) residues 112-124, wasidentified using the program Sciprotein, Scivision (Burlington, Mass.).The following is a synthetic oligonucleotide encoding this peptide andusing the codon bias preferred for expression of nuclear genes in thealga Chlamydomomas reinhardtii: (SEQ ID NO.2)5′-GATCTAGATTAACCTTCAGCTCCTTGGCGGGGCCGTCCTTGTTGTACACGCCCCCACCTTGGTGCGCCGTCAGAG-3′

[0090] The above synthetic oligonucleotide was used to generate a fusiongene between the 3′ end of a low CO₂-induced plasma-membrane proteingene (Genbank accession no. U31976) and the P57 antigen encodingsequence creating by adding the above P57 fragment 3′ of nucleotide 677of U31976, shown below: (SEQ ID NO.:3) 5′-ATGTCGGGCT TGAACAAGTTCATCTATGTG GGCCTCGTTA    TCTCGCAGCT GCTGACTCTG GCGGCCTACG TGGTCGTCAC   GGCCGGCGCT GCCCTTCTGC AGAAGAAGGC GAACACGCTC    ACTCTGTTTG ACACCCAGGAGGGCATTGAC AAGTACACTC    CCGTTTACAA GGAGGTCTTC ACGGCGACCA CCTACATCAT   CGCCTACCCC CAGCAGCCCC AGTACCAGTT CCAGTACCAG    TGGTGGATCA TCCAGTTCGAGCTGTTTGTG TTCCTGCTCA    CCGCCGCCTG CACCGTCTTC CCCTCCATCA TCAAGCGCAT   GCGCCCCGTG GCCCTGACCT TCATCGCCTC CGCCCTGGTG    CTGGTCATGG ACAACATCAACGCCATCTTC TTCCTGCTCC    GCAACGAGAC CGCCAAGGCT GTGTTCGACG ACTACCGCAT   CGCCACCGCT CAGGCTGGCC TGATCATGGT TGGCGTGGCG    AACGGCCTGA CCATCTTCTTCCTGGGCTCG TACGACGCTG    AGGAGTCGCA TGCGATGCCC AACGTGCACG TCACCTCTGA   CGGCGCCACC AAGGTGGGCG GCGTGTACAA CAAGGACGGC    CCCGCCAAGG AGCTGAAGGTGTAA-3′

[0091] It was expected that the P57 epitope would be expressed on theperiplasm side of the cell membrane, as shown in FIG. 3. The gene fusionthen was cloned into the multi-cloning site of plasmid pSSCR7 undercontrol of the β₂ tubulin promoter of Chlamydomonas. pSSCR7 wasconstructed by cloning the HindIII/EcoRI fragment (˜2.7 Kbp) thatcarries the Chlamydomonas β₂-tubulin promoter and the 5′ end ofarysulfatase gene (˜1.0 Kbp) from pJD55 (Davies, 1992 Nucleic AcidsResearch 20: 2959-2965) into pUC18, and designated as pβ₂TU1. In orderto eliminate the 5′ end of arysulfatase gene, the β₂-tubulin promoterwas amplified by PCR. The PCR product was cloned into the BamHI/EcoRIsites of pUC18 to make plasmid pβ₂TU2. The TATA box was found by DNAsequence analysis ˜100 bp away from BamHI site. In order to introduce aunique NdeI site which contains an ATG codon, and a unique NarI site,which was used to clone the 3′ terminator, a NdeI site was removed frompUC18 to make pUN and both sites were removed from pUC18 to make pUNN. AXhoI/Nar fragment containing the 3′-terminator from Chlamydomonas lowCO₂-induced membrane protein gene, as follows: (SEQ ID NO.4)5′-GGCGCCATCT AAGCAGAAGG CTGTGGGATG TGTCACCGTT    AAGCATCGGA GTTTGGGAAGTAGAGAATCT GGGGCTGCGG    TTTTGTGGTT TGCCGCTGCG GTCTGCACTT GGCAGGGTTG   CCCCAGGTCT TGGGGTGACA GTTTAGTTGC TAGGTTGGTA    GCATGTCCTT CGTGACACCAGCGCATTGCA CCCGCTATGT    ACATTCATCG TTTTGGGTCT GGAGCGCTGC GCAGCACCTT   TGGGTAGCGA ATACTTCGGG TGAGCTGCTT ATCTGTATGG    TACGGATGGG CACGGCTCCAAGCAGCAATA CACGGACGCA    CATGCACCAA ATTTTGGTTG TTTGAGTGGA CCGGCTTTAT   CCAACGGTTC AGGTTTGGTT GCTCTCTCCA TCGGAAGCAG    AGCAGAAGCA CAACACACGTCGCAAACATG ATTGGAGCCA    AGGAGCATGA AATGCGAAAG AGCTGGACCA TGCACAGCGC   ATGTAATAAG AGTACTGCAG A-3′

[0092] was combined with β₂-tabulin promoter to form expression vectorpSSCR1. A KpnI/SstI fragment containing the multicloning sites frompBluscript II KS was cloned into pSSCR5 to make the final expressionvector, pSSCR7. (FIG. 1.)

[0093] As seen in FIG. 1, the pSSCR7 has sixteen useful cloning sitesincluding NaeI, KpnI, ApaI, XhoI, SalI, ClaI, HindIII, EcoRV, EcoRI,PstI, SmaI BamHI, SpeI, XbaI, NotI and NarI, that can be used forintroducing one or more coding sequences, as shown in FIG. 1. The fusiongene described above was cloned into the NdeI and XbaI cites.

[0094] The resulting plasmid (pCREpitope) was co-transformed into thenucleus by electroporation into Chlamydomonas reinhardtii strain CC-425using an equimolar amount of p389, a plasmid containing the Arg-7 gene(see Debuchy et al., EMBO, 1989. Vol 8, 2803-2809). The Arg-7 genecompliments the arginine auxotrophic strain of Chlamydomonas, CC-425(Debuchy et al., EMBO, 1989. Vol 8, 2803-2809). Transformants wereobtained after plating the cells on TAP-agar containing 100 μg arginineper ml of medium. The plates were illuminated with fluorescent tubes at10 μmol photons/m²/sec at 22-27° C. Transformants were found after 9-10days of incubation. The resulting transfected algae expressing thefusion protein are known as E-22.

[0095] The main features of the new expression vector are (i) the use ofthe β₂-tubulin promoter, (ii) the construction of sixteen unique cloningsites, (iii) the use of high copy number (in E. coli), pUC18, as theoriginal plasmid, and (iv) the small size of new expression vector (4.4Kpb).

[0096] Another plasmid was created by inserting the entire P57 gene intopCPPTG, as shown in FIG. 2. pCPPTG is a plasmid constructed forexpression of foreign genes in chloroplasts. It based on a modified E.coli vector, pUC18. As seen in FIG. 2, pCPPTG comprises the psbApromoter, psbA terminator, aadA cassette, and a multicloning sitelocated between the psbA promoter and terminator. The psbA promoter,psbA terminator, and aadA cassette are derived from the pBA155dH3plasmid, while the multicloning site is derived from pBluescript IIKS(described in Hutchison et al, (1996) Chapter 9, and Ruffle et al,Chapter 16; In Molecular Genetics of Photosynthesis, Frontiers inMolecular Biology. Oxford Univ. Press). The entire P57 gene from R.salmoninarum was cloned into the multicloning site, specifically theApaI and PmeI sites, of pCPPTG.

[0097] Co-transformants growing on medium lacking arginine andcontaining the membrane protein-P57 fusion were identified by PCRamplification of the membrane protein-P57 fusion gene using thefollowing oligonucleotide primers: Primer C113-5′-AGCATATGGGGCCCATGTCGGGCTTGAACAAGTTCATCT (SEQ ID NO.5) EPITOPE-3′-GATCTAGATTAACCTTCAGCTCCTTGGCGGGGCCGT (SEQ ID NO.6)CCTTGTTGTACACGCCCCCACCTTGGTGCGCCGTCAGAG

[0098] In order to perform PCR on the transformants, total genomic DNAfrom C. reinhardtii was isolated. To do this, cell cultures (20 ml) werepelleted by certification and resuspended in 0.35 ml TEN buffer. Theresuspended cells were incubated with 50 μl of 2 mg/ml proteinase K and25 μl of 20% SDS for 2 hr at 55° C. Then, 50 μl of 5M potassium acetatewas added and the cells were incubated on ice for 30 min. The lysate wasextracted by phenol:chloroform and DNA was precipitated by ethanol.

[0099] Those cells that were positive for the presence of the membraneprotein-P57 fusion product by PCR were then were screened by westernblot analysis using antibodies against the intact P57 protein. Asdemonstrated by western blot analysis (see FIG. 5), antibodies generatedagainst the intact P57 protein recognized the 57 kD P57 protein fromsolubilized whole cell extracts of transgenic algae (CP57) expressingthe P57 protein. No protein was detected on the western blot fornon-transformed cells.

Example 2 Immersion

[0100] Transgenic Chlamydomonas reinhardtii expressing the P57 proteinfrom Rennibacterium salmoninarum as a fusion protein on the plasmamembrane (called E-22 algae) or in the chloroplast (called CP57 algae)were constructed as described in Example 1. Control algae (the CC-2137strain of C. reinhardtii) were also used to test administration byimmerson.

[0101] The algae were grown in tris-acetate-phosphate (TAP) medium. SeeGorman D S and Levine R P (1965), Proc. Nat. Acad. Sci. 54: 1665-1669,to a density of 1×10⁶ cells/ml at 22-27° C. with 10 μmol photons/m²/secillumination from fluorescent tubes. The algal cells were harvested bycentrifugation and resuspended in water to a density of approximately1×10⁶ cells/ml.

[0102] Rainbow trout juveniles (average initial weight, 9.1±0.5 g) weresubjected to a bath treatment in water containing algae in a 20 L barrelcontainer (one barrel containing each of the E-22, CP57 and CC-744algae). Exposure of the trout to the various algae was for 2 hours withintense aeration of the water. The concentrations of the three types ofalgae were as follows: CP57, 7.97×10⁵ cells/ml; 2137, 2.71×10⁶ cells/ml;and E-22, 2.97 10⁶ cells/ml. A control treatment was also performedwhere additional fish were handled identically, except that they werenot exposed to algae (sham inmmersion). After immersion, both controlsand those immersed in algae, were distributed into 3 tanks per immersiontreatment. All the fish groups were fed with the same commercial diet atthe rate of 2% of fish weight for 3 weeks (Bioproducts, Inc. Oreg.).After that period, the fish were immersed again in a 2^(nd) treatmentaccording to the same procedure as in the first treatment. Theconcentrations of the three algae types for the 2^(nd) treatment were asfollows: CP57, 1.46×10⁶ cells/ml; 2137, 6.57×10⁵ cells/ml; and E-22,1.23×10⁶ cells/ml. The fish were fed with the commercial diet for 4weeks until the second sampling in the experiment (7 weeks) (a firstsampling was performed prior to any feeding). All fish were fasted for24 h prior to treatment.

[0103] The water temperature increased gradually from 12 to 17° C.during the course experiment due to seasonal environmental changes.Diurnal light:dark cycle was regulated at 12 h: 12 h. Total fish weightin each tank was measured 3 weeks after the first treatment, and then at7 and 9 weeks after the first immunization. The fish were kept for thelast 2 weeks at the feeding rate of 1.5% of fish weight.

[0104] Blood was taken before the initiation of the experiment and atthe time of weighing fish (3, 7, and 9 weeks) from caudal vein withheparinized syringe for plasma, and with non-heparinized syringe forserum. Six fish were randomly selected per tank for the determinationsof hematocrit, hemoglobin, liver and spleen weights. Mucus from the fishwas collected and frozen. Hematocrit was determined by themicrohematocrit method. Total hemoglobin was determined with SigmaDiagnostic Kits procedure No. 525) by using human hemoglobin solution asstandard. All procedures and handling of animals were conducted incompliance with the guidelines of the Institutional Laboratory AnimalCare and Use Committee, The Ohio State University. TABLE 1 Weight,Spleen Relative Weight (SRW), Hematocrit and Hemoglobin of Fish Treatedby Immersion Final weight SRW¹ Hematocrit Hemoglobin Treatment (mean ±SD) (% body weight) (%) (g/100 mlk) Control 22.0 ± 1.90 0.080 ±0.015^(ab) 37.8 ± 3.51 8.47 ± 1.09 E-22 22.4 ± 1.36 0.082 ± 0.012^(a )36.2 ± 2.57 8.39 ± 0.85 CP57 21.5 ± 1.14 0.097 ± 0.004^(a ) 36.5 ± 1.507.66 ± 0.31 2137 21.8 ± 1.34 0.082 ± 0.004^(a ) 35.2 ± 2.25 7.84 ± 0.77

[0105] The fish growth and physiological parameter values in the tableabove show that there were no significant differences in growth rate(final weight), hematocrit, and hemoglobin among all the treatmentgroups (P>0.05).

Example 3 Feed Pellet

[0106] Transgenic Chlamydomonas reinhardtii expressing the P57 proteinfrom Rennibacterium salmoninarum as a fusion protein on the plasmamembrane (called E-22 algae) or in the chloroplast (called CP57 algae)were prepared as described in Example 1.

[0107] The algae were grown in TAP medium as described above in Example2 to a density of 1×10⁶ cells/ml. The algal cells were harvested bycentrifugation, frozen in liquid nitrogen and freeze-dried.

[0108] Three semi-purified diets formulated based on casein-gelatin as aprotein source were used for oral administration. The threesemi-purified diets were isonitrogenous and isocaloric to incorporate 4%(on dry weight basis) of three different types of algae. The three algaewere E-22, CP57, and CC-2137. Five percent of fish protein concentrate(CPSP 90, Sopropeche S. A., Boulogne-Sur-Mer, France) was supplementedinto the diets to enhance their palatability. The dietary ingredientswere mixed with distilled water and cold-pelleted into 2.0 mm diametersize, and then freeze-dried to have less than 5% moisture. Diets werecrushed and sieved into a desirable particle size (0.8-2.0 mm), andstored at −20° C. until use.

[0109] Rainbow trout juveniles (average initial weight, 9.1±0.5 g) wererandomly distributed into 24 rectangular tanks (20 L capacity) at adensity of 22 fish per tank, 3 tanks per dietary treatment. A controlgroup was also used (each in triplicate tanks) and were fed a commercialdiet (Bioproducts, Inc. Oreg.). The control group was not exposed to anydisturbances. Each experimental diet was fed to a group (3 tanks) offish at the feeding rate of 2% of fish body weight for 3 consecutivedays. Accordingly, the results in an approximate intake of 25 mg ofalgae protein/100 g fish body weight/day. After 3 days of feeding algaediets twice a day, all the fish groups were fed with the same commercialdiet for 3 weeks (2.5% per day), twice per day, 7 days per week. The2^(nd) oral treatment (boost) was conducted again with the same algaediets and the commercial diet at the feeding rate of 2% of fish bodyweight for 4 days. After the 4 days of the 2^(nd) oral treatment, allthe fish groups were fed the same commercial diet for 4 weeks until thesecond sampling in this experiment. The fish were kept for 2 additionalweeks at the feeding rate of 1.5% of fish weight and sampled again(third sampling; 9 weeks). TABLE 2 Weight, Spleen Relative Weight (SRW),Hematocrit and Hemoglobin of Fish Treated Orally Final weight SRW¹Hematocrit Hemoglobin Treatment (mean ± SD) (% body weight) (%) (g/100mlk) Control 19.9 ± 0.60 0.093 ± 0.004^(a ) 38.3 ± 5.69 9.52 ± 0.96 E-2221.5 ± 1.00 0.053 ± 0.001^(c ) 38.5 ± 1.80 8.40 ± 0.83 CP57 22.0 ± 0.120.056 ± 0.004^(bc) 39.5 ± 3.12 8.34 ± 0.62 2137 20.6 ± 0.66 0.052 ±0.003^(c ) 35.3 ± 0.58 7.61 ± 0.02

[0110] The fish growth and physiological parameter values in the ableabove show that there were no significant differences in growth rate(final weight), hematocrit, and hemoglobin among all the treatmentgroups (P>0.05). Spleen relative weight (SRW) was significantly lower inorally treated fish groups than in a control fish group and all the fishgroups in immersion treatments (P<0.05).

Example 4 Serum and Mucus Antibodies in Fish After Administration ofAlgae

[0111] Serum and mucus obtained from the fish treated by the immersionmethod (Example 2) as well as the oral feeding method (Example 3) wereexamined for the presence of antibodies reactive with the P57 immunogensexpressed by the transgenic algae. This was done by using the serum orthe mucus from fish fed CP57, E-22, CC-2137, or no algae to probewestern blot membranes to which were transferred proteins from SDS-PAGEgels of solubilized wild-type Chlamydomonas cells or transgenicChlamydomonas expressing the P57 protein in the chloroplast (CP57), oras a fusion protein between the high CO₂ induced protein and the P57antigenic determinant (E-22).

[0112] Wild-type, E-22 and CP57 algae were boiled in SDS-PAGE loadingbuffer for 5 minutes. Samples were loaded at 15 μg chlorophyll per well(see Arnon D (1949), Plant Physiol. 24: 1-15, for additional informationon chlorophyll assays) on a 12.5% acrylamide gel with a 6% acrylamidestacking gel. The samples were electrophoresed at 15-18 mAmp constantcurrent for 5-6 hr. Following electrophoresis the proteins weretransferred from the gel to immobilon-P (PVDF=polyvinylidene fluoride)membrane using a semi-dry system at 1.25 mA per square centimeter ofmembrane for 2-3 hr. After removing the membrane from semi-dryelectroblotter, the membrane was soaked in methanol for 15 second anddried at room temperature for 30 minutes. The membrane was then washedtwice with PBST buffer (10 mM Sodium phosphate, 150 mM NaCl, 1%Tween-20, pH 7.4) for 5 minute each. (0.25 mL PBST per square centimeterof membrane) and blocked with 3% casein (in PBST buffer) at 0.25 mL persquare centimeter for 1-2 hr. The membrane was then washed twice withPBST buffer (10 mM Sodium phosphate, 150 mM NaCl, 1% Tween-20, pH 7.4)for 5 minute each. (0.25 mM PBST per square centimeter membrane).

[0113] All fish mucus and serum were treated with soluble protein fromCC-425 algal strain at 1:2 (vol/vol ratio) for 1 hr at room temperaturefollowed by centrifugation to remove non-specifically bound proteins.Fish mucus or sera was used at a 1:150 dilution in 1% BSA in PBST andincubated at room temperature for 2 hr (used 0.1 mL of serum or mucussolution in buffer per square centimeter of blotting membrane). Themembrane was then washed twice with PBST buffer (10 mM sodium phosphate,150 mM NaCl, 1% Tween-20, pH 7.4) for 5 minute each (0.25 mL PBST persquare centimeter of membrane). This was followed by incubation withmouse anti IgM Rainbow trout serum (1:200 dilution in 1% bovine serumalbumin (BSA) in PBST) at room temperature for 2 hr (0.1 mL dilutionbuffer per square centimeter of membrane). The membrane was then washedtwice with PBST buffer for 5 minute each (0.25 mL PBST per squarecentimeter of membrane) followed by incubation with goat anti-mouseantisera conjugated to horseradish peroxidase (HRP) at a 1:3,000dilution in 1% BSA in PBST at room temperature for 1 hr (0.25 mLdilution buffer per square centimeter of membrane). The membrane wasthen washed twice with PBST buffer for 5 minute each. (0.25 mL PBST persquare centimeter of membrane). The HRP detection system was Opti-4CNSubstrate Kit from Bio-Rad. Opti-4CN is an improved and more sensitiveversion of the calorimetric horseradish peroxidase (HRP) substrate,4-chloro-1-naphthol (4CN). Normally, this step takes time about 20-30minutes (0.25 mL substrate per square centimeter of membrane).

[0114] The results of the studies are shown in FIG. 4 which shows serafrom fish that were fed algae in their diet, and in FIG. 5 which showsmucus from fish that were immersed in algae.

[0115] The first western blot (FIG. 4) shows that sera from fish fedpellets containing either E-22 or CP57 algae recognized a 57 kD proteinpresent in algae (CP57) expressing the P57 protein in the chloroplast(lanes 2 and 5). Significantly, no 57 kD band was detected when usingsera from fish fed either wild type or no algae in the diet. Inaddition, two protein bands were detected at 22 and 27 kD in all serumtreatments. These bands arise from non-specific interactions. Incontrast, no 57 KD proteins were detected in serum from fish that wereimmersed in either E-22, CP57, wild-type or no algae. These resultsindicate that fish fed food containing transgenic algae expressing theP57 protein of Rennibacterium salmoninarum generated specific antibodiesagainst the P57 protein. Unfortunately, due the cross-contaminating bandat 22 kD) it was not possible to determine whether fish fed pelletscontaining E-22 algae generated antibodies against the 22 kD fusionprotein. However, the fact that the P57 protein present in CP57 algaewas detected using sera from E-22 fed fish suggests that the 22 kDfusion protein was immunogenic against the P57 antigenic determinantpresent in the fusion protein.

[0116] In addition, fish mucus was tested for the presence of antibodiesgenerated against the P57 protein. As shown in FIG. 5, western blotsprobed with mucus from fish immersed in E-22 and CP57 algae wasimmunoreactive against the P57 protein present in CP57 algae (lanes 2and 5). Again no immune reaction was observed using mucus from fishimmersed in wild-type or no algae. These results demonstrate thatP57-specific antibodies were expressed in mucus of fish immersed in E-22and/or CP57 algae. There also appears to be an immune reaction specificfor the 22 kD fusion protein when using mucus from fish immersed in E-22algae (lane 1). However, this result is tentative since a non-specificinteraction is observed at the same molecular weight in lanes 5 and 6.Significantly, immersion of fish with any algal strain failed togenerate P57-specific antibodies in sera. Overall, these resultsindicate that immersion with algae expressing foreign and immunogenicproteins (P57) is an effective means to generate the production ofprotective antibodies in the mucus of fish.

Example 5 Algae Expressing WSSV Proteins and Administration to Shrimp

[0117] White Spot Syndrome Virus (WSSV) is a cause of disease of shrimp.Shrimp production losses of 80% due to WSSV infection have been reportedand shrimp farms can be shut down for periods of up to two yearsfollowing a WSSV infestation.

[0118] VP28, VP26, VP24, and VP19 are known proteins from WSSV (PCT Int.Appl. No. WO 0109340). Using gene specific primers, each of the fourknown viral proteins is amplified using WSSV DNA as a template (vanHulten, et al., 2001, PCT International Publication WO 0109340). Each ofthese genes is cloned separately and together into a Chlamydomonasexpression vector similar to the methods described in Example 1. Forexample the pSSCR7 plasmid which drives high-level expression in thecytoplasm, and the pSSCR7 vector having a 5′ aryl sulfatase periplasmictargeting transit sequence (Davies et al., 1994, Plant Cell 6: 53-63).The aforementioned vectors randomly integrate into the Chlamydomonasnuclear genome. In addition, a proprietary chloroplast transformationvector that integrates into the chloroplast genome by homologousrecombination can be used (Hutchison, et al., 1996, Chapter 9,Chloroplast transformation. Pgs. 180-196; In: Molecular Genetics ofPhotosynthesis, Frontiers in Molecular Biology. Anderson B., Salter, AH, and Barber J. eds.; Oxford University Press). Each of these vectorscontains promoters that drive high levels of expression in the cytoplasmor chloroplast. Viral protein expression is quantified by western blotanalysis normalized against known loadings of WSSV (see objective 1A4for quantification of WSSV). For the western blot analysis polyclonalantibodies are generated against WSSV proteins by injection of purifiedand heat-denatured WSSV into rabbits.

[0119] The various expression systems are used to determine whichpattern of expression (periplasmic, cytoplasmic or chloroplast) mosteffectively induces the shrimp “immune-response” (Bachere, 2000,Aquaculture 191: 3-11; Rombout et al., 1985, Cell Tissue Res. 239:519-30). While periplasmic expressed viral proteins are expected mosteffective, they are potentially most vulnerable to digestion cytoplasmicand chloroplast expressed proteins would be progressively lesssusceptible to digestion (D'Souza, et al., 1999, Marine Biol. 133:621-633).

[0120] PSF (pathogenic specific free) shrimp larvae are fed eitherwild-type or VP-expressing microalgae 3-5 days prior to challenge withknown titers of WSSV. During the WSSV challenge, the shrimp larvae arefed the appropriate algal strain either as live algae or as dried algaein feed pellets (D'Souza, et al., 1999, Marine Biol. 133: 621-633).Various concentrations of algae feed are compared. Relative percentsurvival will be calculated during the weeks following WSSV exposure forall treatments. As described below, molecular markers specific forshrimp disease inducible genes will be used in quantitative realtime-PCR experiments to evaluate the “immune” response in shrimp priorto and after exposure to 1) WSSV VP expressing algae, 2) wild typealgae, 3) WSSV VP expressing algae followed by WSSV exposure, 4) WSSValone, and 4) no algae or WSSV. Based on studies with injected WSSVproteins (van Hulten, et al., 2001, PCT International Publication WO0109340) it is expected that shrimp fed microalgae expressing WSSV VPproteins will be protected from WSSV infection.

Example 6 Rabbits

[0121] Transgenic algae expressing antigenic proteins (in thechloroplast, cytoplasm, or on the cell surface as fusion proteins) aredelivered to rabbits as a component of the feed pellets, essentially asdescribed above for fish. Alternatively, the algae are delivered asmixed with the drinking water. The transgenic algae may, for example,express the CS6 and Vi antigens of Salmonella typhi.

[0122] Although the invention has been described in detail withreference to certain preferred embodiments, variations and modificationsexist within the scope and spirit of the invention as described anddefined in the following claims.

1 6 1 13 PRT Renibacterium salmoninarum 1 Val Tyr Asn Lys Asp Gly ProAla Lys Glu Leu Lys Val 1 5 10 2 75 DNA Renibacterium salmoninarum 2gatctagatt aaccttcagc tccttggcgg ggccgtcctt gttgtacacg cccccacctt 60ggtgcgccgt cagag 75 3 624 DNA Chlamydomonas reinhardtii 3 atgtcgggcttgaacaagtt catctatgtg ggcctcgtta tctcgcagct gctgactctg 60 gcggcctacgtggtcgtcac ggccggcgct gcccttctgc agaagaaggc gaacacgctc 120 actctgtttgacacccagga gggcattgac aagtacactc ccgtttacaa ggaggtcttc 180 acggcgaccacctacatcat cgcctacccc cagcagcccc agtaccagtt ccagtaccag 240 tggtggatcatccagttcga gctgtttgtg ttcctgctga ccgccgcctg caccgtcttc 300 ccctccatcatcaagcgcat gcgccccgtg gccctgacct tcatcgcctc cgccctggtg 360 ctggtcatggacaacatcaa cgccatcttc ttcctgctcc gcaacgagac cgccaaggct 420 gtgttcgacgactaccgcat cgccaccgct caggctggcc tgatcatggt tggcgtggcg 480 aacggcctgaccatcttctt cctgggctcg tacgacgctg aggagtcgca tgcgatgccc 540 aacgtgcacgtcacctctga cggcgccacc aaggtgggcg gcgtgtacaa caaggacggc 600 cccgccaaggagctgaaggt gtaa 624 4 501 DNA Chlamydomonas reinhardtii 4 ggcgccatctaagcagaagg ctgtgggatg tgtcaccgtt aagcatcgga gtttgggaag 60 tagagaatctggggctgcgg ttttgtggtt tgccgctgcg gtctgcactt ggcagggttg 120 ccccaggtcttggggtgaca gtttagttgc taggttggta gcatgtcctt cgtgacacca 180 gcgcattgcacccgctatgt acattcatcg ttttgggtct ggagcgctgc gcagcacctt 240 tgggtagcgaatacttcggg tgagctgctt atctgtatgg tacggatggg cacggctcca 300 agcagcaatacacggacgca catgcaccaa attttggttg tttgagtgga ccggctttat 360 ccaacggttcaggtttggtt gctctctcca tcggaagcag agcagaagca caacacacgt 420 cgcaaacatgattggagcca aggagcatga aatgcgaaag agctggacca tgcacagcgc 480 atgtaataagagtactgcag a 501 5 39 DNA Artificial Sequence synthetic primer for PCR 5agcatatggg gcccatgtcg ggcttgaaca agttcatct 39 6 75 DNA ArtificialSequence synthetic primer for PCR 6 gatctagatt aaccttcagc tccttggcggggccgtcctt gttgtacacg cccccacctt 60 ggtgcgccgt cagag 75

1. A delivery system for delivering a biologically active protein to ahost animal comprising an algal cell transformed by an expressionvector, and the expression vector comprising a nucleotide sequencecoding for the biologically active protein, operably linked to apromoter.
 2. The delivery system of claim 1, wherein the algal cell issuspended in water for immersing the host animal.
 3. The delivery systemof claim 1 wherein the biologically active protein is selected from thegroup consisting of hormones and antimicrobial peptides.
 4. The deliverysystem of claim 1 wherein the algal cell is mixed with a sample of feedfor the host animal.
 5. The delivery system of claim 1 wherein theanimal is selected from the group consisting of mammals, fish, birds,and crustaceans.
 6. The delivery system of claim 1 wherein thebiologically active protein is a peptide derived from the groupconsisting of bactericidal proteins, insecticidal proteins, growthhormones, and antigens.
 7. A delivery system for delivering antigens toa host animal comprising an algal cell transformed by an expressionvector, and the expression vector comprising a nucleotide sequencecoding for an antigenic determinant.
 8. The delivery system of claim 7,wherein the expression vector further comprises a promoter operablylinked to the nucleotide sequence coding for the antigenic determinant.9. The delivery system of claim 8, wherein the expression vector furthercomprises a terminator for terminating transcription.
 10. The deliverysystem of claim 9, wherein the algal cell expresses the antigenicdeterminant in an area selected from the group consisting of nucleus,chloroplast, mitochondria, periplasmic space, cell membrane, or cellwall.
 11. The delivery system of claim 7, wherein the algal cell isselected from the group consisting of green algae, brown algae, ordiatoms.
 12. The delivery system of claim 7 wherein the algal cell isChlamydomonas reinhardtii.
 13. The delivery system of claim 7 whereinthe antigenic determinant is a holoprotein or protein fragment from apathogenic organism in the host animal.
 14. The delivery system of claim13 wherein the antigenic determinant is part of a fusion protein. 15.The delivery system of claim 7 wherein the algal cell is disabled. 16.The delivery system of claim 7 wherein the algal cell is packaged as afood product.
 17. The delivery system of claim 16 wherein the foodproduct is a capsule.
 18. The delivery system of claim 7 whereinmultiple algal cells are incorporated into a pellet.
 19. The deliverysystem of claim 18 wherein the algal cells are selected from the groupconsisting of living and dead cells.
 20. The delivery system of claim 7wherein the algal cell is suspended in water for immersing the hostanimal.
 21. The delivery system of claim 20 wherein the algal cell isdried and formed into a powder prior to being suspended in water. 22.The delivery system of claim 7 wherein the algal cell is mixed with aliquid and packaged as a drink.
 23. The delivery system of claim 7wherein the expression vector is incorporated into genetic materialselected from the group consisting of nuclear, chloroplast, andmitochondrial.
 24. A method for inducing an immune response in ananimal, comprising the steps of obtaining a transgenic alga expressingan antigenic peptide, and administering the transgenic alga to theanimal.
 25. The method of claim 24 wherein the transgenic alga isadministered by feeding.
 26. The method of claim 25 wherein the animalis a fish, and the immune response is detectable in a sample of serumfrom the fish.
 27. The method of claim 24 wherein the transgenic alga isadministered by immersing the animal in a suspension comprising thealga.
 28. The method of claim 27 wherein the animal is a fish, and theimmune response is detectable in a sample of mucus from the fish.
 29. Amethod for inducing enhanced growth in an animal, comprising the stepsof obtaining a transgenic alga expressing a peptide derived from agrowth hormone, and administering the transgenic alga to the animal. 30.A method for controlling a pathogenic population in an animal,comprising the steps of obtaining a transgenic alga expressing a peptidederived from a bactericidal or insecticidal protein, and administeringthe transgenic alga to the animal.